Method and apparatus for performing an assay

Abstract

This invention provides apparatus and methods that enable the direct measurement of an analyte sample in an assay well by forming an independent measuring region. The sample to be measured can be separated into portions, for example, a bound portion and a free portion. To form the independent measuring region, a layer-forming material that can restrict the passage of radiant energy that is measured by a detection device is added to the assay well. The layer-forming material forms a layer that restricts the passage of radiant energy from the region. Thus, a sample portion in one separate region may be measured by a detection device separately and independently of a portion of the sample in a different region. In one example, a second portion in another region can be separately and independently measured by a detection device without the removal of the portions from the assay well.

I claim:  
     
         1 . A method of performing an assay, the method comprising: 
 providing a sample that comprises a detectable label, wherein the sample is present in an assay vessel and at least a portion of the detectable label becomes associated with a surface of assay vessel;    adding to the sample a layer-forming material that forms a barrier between detectable label that is associated with the surface of the vessel and, if present, detectable label that is not associated the surface of the vessel; and    determining the amount of detectable label that is associated with the surface or that is not associated with the surface.    
     
     
         2 . The method of  claim 1 , wherein the method comprises determining both surface-associated and surface-unassociated detectable label.  
     
     
         3 . The method of  claim 1 , wherein the layer-forming material is opaque to radiant energy.  
     
     
         4 . The method of  claim 3 , wherein the detectable label comprises a radiolabel and the amount of detectable label determined by adding to the sample a liquid scintillent and measuring the luminescence resulting from the interaction of the radiolabel and the scintillent.  
     
     
         5 . The method of  claim 3 , wherein the detectable label emits a luminescent or fluorescent signal.  
     
     
         6 . The method of  claim 5 , wherein the label comprises a luciferase, a fluorescent protein, an enzyme, a fluorescent dye, a semiconductor nanocrystal, or a calorimetric label.  
     
     
         7 . The method of  claim 1 , wherein the detectable label becomes associated with the surface by binding to an analyte that binds to the surface.  
     
     
         8 . The method of  claim 7 , wherein the analyte binds to the surface through a binding moiety that is attached to the surface.  
     
     
         9 . The method of  claim 7 , wherein the detectable label is covalently attached to the analyte.  
     
     
         10 . The method of  claim 7 , wherein the detectable label is indirectly attached to the analyte.  
     
     
         11 . The method of  claim 10 , wherein the detectable label is attached to a labeling moiety that binds to the analyte.  
     
     
         12 . The method of  claim 11 , wherein the labeling moiety comprises an antibody.  
     
     
         13 . The method of  claim 1 , wherein the detectable label is present in a cell that becomes attached to the surface of the reaction vessel.  
     
     
         14 . The method of  claim 13 , wherein the detectable label is present in periplasm or cytoplasm of the cell.  
     
     
         15 . The method of  claim 13 , wherein the detectable label is present on a surface of the cell.  
     
     
         16 . The method of  claim 1 , wherein the layer-forming material forms a barrier by settling onto the bottom surface of the reaction vessel.  
     
     
         17 . The method of  claim 16 , wherein the settling occurs by gravity or by centrifugation.  
     
     
         18 . The method of  claim 1 , wherein the layer-forming material is a paramagnetic particle.  
     
     
         19 . The method of  claim 18 , wherein the method further comprises removing the paramagnetic material using a magnetic field.  
     
     
         20 . An apparatus for performing an assay on a sample in an assay well, comprising: 
 means for adding to the sample a layer-forming material, thereby separating the sample into a first region and a second region; and    means for measuring a parameter of either or both of the first and second region.    
     
     
         21 . The apparatus according to  claim 20 , further comprising means for forming the layer-forming material into a layer.  
     
     
         22 . The apparatus according to  claim 20 , further comprising a means for measuring a parameter of the first region separately from a parameter of the second region.  
     
     
         23 . The apparatus according to  claim 22 , wherein the means for measuring the parameter of the first region substantially concurrently with measuring the parameter of the second region.  
     
     
         24 . The apparatus according to  claim 22 , further including means for calculating a relationship between the parameter measured from the first region and the parameter measured from the second region.  
     
     
         25 . The apparatus according to  claim 20 , wherein the layer-forming material is an opaque material that restricts the passage of radiant energy from one of the first and second regions to the other.